Previously, in studies involving a semi-homologous system of gnotobiotic newborn pigs and a virulent porcine rotavirus strain (SB-1A) and an avirulent human rotavirus strain (DS-1) and their reassortants, we demonstrated that: (i) the third (VP3), fourth (VP4), ninth (VP7), or tenth (NSP4) porcine rotavirus gene each play an important independent role in the virulence of rotavirus infection in piglets; and (ii) all four of the porcine rotavirus virulence-associated genes are required for the induction of diarrhea and the shedding of rotavirus by piglets. These observations suggested a potential new strategy for attenuation of wild-type human rotaviruses of major epidemiologic importance and its application to the development of a safe and effective vaccine. Previously, we developed a rhesus (RRV)- or bovine (UK)-based quadrivalent vaccine which was designed to provide antigenic coverage for VP7 (G) serotypes 1-4 of epidemiologic importance. Last year, we reported (i) construction of eleven porcine rotavirus Gottfried-bsased single VP4 or VP7 gene substitution reassortant vaccine candidates. This year, in order to study which gene(s) of a murine rotavirus EB strain are involved in induction of diarrhea in a mouse model, we passed serially cell culture adapted avirulent EB strain in mouse pups. After six or seven serial in vivo passages in mouse pups, the cell culture adapted avirulent EB strain became diarrheagenic in infected pups. We are in the middle of sequencing 11 genes of both avirulent and virulent EB strains to identify which gene(s) are involved in this phenomenon.